Proteolytic processing of MucA protein in SOS mutagenesis: both processed and unprocessed MucA may be active in the mutagenesis.
The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ'-'MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA+ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.
Shiba
T
T
Department of Experimental Chemotherapy, Osaka University, Japan.
Iwasaki
H
H
Nakata
A
A
Shinagawa
H
H
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D010447:Peptide Hydrolases / Q000235:genetics; D010957:Plasmids; D015183:Restriction Mapping; D013014:SOS Response, Genetics; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; C063757:MucA protein, Bacteria; D010447:Peptide Hydrolases